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Molecular and antigenic characteristics of Massachusetts genotype infectious bronchitis coronavirus in China Lingfeng Chen, Tingting Zhang, Zongxi Han, Shuling Liang, Yang Xu, Qianqian Xu, Yuqiu Chen, Yan Zhao, Yuhao Shao, Huixin Li, Kexiong Wang, Xiangang Kong, Shengwang Liu* Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, Peoples Republic of China A R T I C L E I N F O Article history: Received 16 August 2015 Received in revised form 7 October 2015 Accepted 7 October 2015 Keywords: Infectious bronchitis coronavirus Massachusetts type Recombination event Serotype Vaccination-challenge test A B S T R A C T In this study, 418 IBVs were isolated in samples from 1717 chicken ocks. Twenty-nine of the isolates were classied as the Massachusetts genotype. These 29 isolates, as well as two previously isolated Massachusetts genotype IBV strains, were studied further. Of the 31 strains, 24 were H120-like and two were M41-like isolates as determined by complete genomic sequence analysis, indicating that most of the IBV isolates were likely the reisolated vaccine virus. The remaining ve IBV isolates, ck/CH/LHB/111172, ck/CH/LSD/111219, ck/CH/LHB/130598, ck/CH/LDL/110931, and ck/CH/LHB/130573, were shown to have originated from natural recombination events between an H120-like vaccine strain and other types of viruses. The virus cross-neutralization test found that the antigenicity of ck/CH/LHB/111172, ck/CH/LSD/ 111219, and ck/CH/LHB/130598 was similar to that of H120. Vaccination with the H120 vaccine offered complete protection against challenge with these isolates. However, isolates ck/CH/LDL/110931 and ck/ CH/LHB/130573 were serotypically different from their parental viruses and from other serotypes in this study. Furthermore, vaccination with the H120 vaccine did not provide protection against challenge with these two isolates. The results of this study demonstrated that recombination is the mechanism that is responsible for the emergence of new serotype strains, and it has the ability to alter virus serotypes. Therefore, IBV surveillance of chicken ocks vaccinated with IBV live vaccines, as well as the consideration of new strategies to effectively control IBV infection using inactivated or/and genetically engineered vaccines, is of great importance. ã 2015 Elsevier B.V. All rights reserved. 1. Introduction Avian infectious bronchitis virus (IBV) is a ubiquitous, highly contagious respiratory pathogen of chickens that inicts serious economic losses to the commercial poultry industry worldwide. IBV belongs to the genus Gammacoronavirus in the subfamily Coronavirinae, family Coronaviridae, order Nidovirales (de Groot et al., 2012). It has a single-stranded, positive-sense RNA genome of approximately 27.6 kilobases in length that encodes four structural proteins: the nucleocapsid (N), membrane (M), envelope (E), and spike (S). Serotype and genotype classications, which are usually based on features of the S1 part of the S protein gene, are used to classify IBV strains (de Wit, 2000). Furthermore, recombination may be involved in the evolution of IBV, and it probably occurs at many positions within a given genome during mixed infections (Lai and Cavanagh, 1997). Historically, the Massachusetts (Mass)-type viruses were believed to be the rst and only serotype found in the USA and other regions of the world. However, many IBV serotypes have arisen and disappeared in the poultry industry since then (Cook et al., 2012). The control of IB is mostly accomplished through the use of live attenuated vaccines. The use of commercially produced Mass-type modied live virus vaccines began in the 1950s and has continued to this day. This type of vaccines, such as the Mass strains M41 and H120, are the most commonly used around the world because such vaccines have proven to be capable of protecting against a wide range of IBV strains (Cavanagh and Gelb, 2008). However, the Mass-type of IBVs is constantly isolated from chicken ocks with respiratory clinical signs. * Corresponding author at: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin 150001, Peoples Republic of China. Fax: +86 451 82734181. E-mail address: [email protected] (S. Liu). http://dx.doi.org/10.1016/j.vetmic.2015.10.003 0378-1135/ ã 2015 Elsevier B.V. All rights reserved. Veterinary Microbiology 181 (2015) 241251 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.else vie r.com/locate /ve tmic

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Page 1: 2015 Molecular and antigenic characteristics of Massachusetts genotype infectious bronchitis coronavirus in China

Veterinary Microbiology 181 (2015) 241–251

Molecular and antigenic characteristics of Massachusetts genotypeinfectious bronchitis coronavirus in China

Lingfeng Chen, Tingting Zhang, Zongxi Han, Shuling Liang, Yang Xu, Qianqian Xu,Yuqiu Chen, Yan Zhao, Yuhao Shao, Huixin Li, Kexiong Wang, Xiangang Kong,Shengwang Liu*Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy ofAgricultural Sciences, Harbin 150001, People’s Republic of China

A R T I C L E I N F O

Article history:Received 16 August 2015Received in revised form 7 October 2015Accepted 7 October 2015

Keywords:Infectious bronchitis coronavirusMassachusetts typeRecombination eventSerotypeVaccination-challenge test

A B S T R A C T

In this study, 418 IBVs were isolated in samples from 1717 chicken flocks. Twenty-nine of the isolateswere classified as the Massachusetts genotype. These 29 isolates, as well as two previously isolatedMassachusetts genotype IBV strains, were studied further. Of the 31 strains, 24 were H120-like and twowere M41-like isolates as determined by complete genomic sequence analysis, indicating that most of theIBV isolates were likely the reisolated vaccine virus. The remaining five IBV isolates, ck/CH/LHB/111172,ck/CH/LSD/111219, ck/CH/LHB/130598, ck/CH/LDL/110931, and ck/CH/LHB/130573, were shown to haveoriginated from natural recombination events between an H120-like vaccine strain and other types ofviruses. The virus cross-neutralization test found that the antigenicity of ck/CH/LHB/111172, ck/CH/LSD/111219, and ck/CH/LHB/130598 was similar to that of H120. Vaccination with the H120 vaccine offeredcomplete protection against challenge with these isolates. However, isolates ck/CH/LDL/110931 and ck/CH/LHB/130573 were serotypically different from their parental viruses and from other serotypes in thisstudy. Furthermore, vaccination with the H120 vaccine did not provide protection against challenge withthese two isolates. The results of this study demonstrated that recombination is the mechanism that isresponsible for the emergence of new serotype strains, and it has the ability to alter virus serotypes.Therefore, IBV surveillance of chicken flocks vaccinated with IBV live vaccines, as well as theconsideration of new strategies to effectively control IBV infection using inactivated or/and geneticallyengineered vaccines, is of great importance.

ã 2015 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Veterinary Microbiology

journal homepage: www.else vie r .com/locate /ve tmic

1. Introduction

Avian infectious bronchitis virus (IBV) is a ubiquitous, highlycontagious respiratory pathogen of chickens that inflicts seriouseconomic losses to the commercial poultry industry worldwide.IBV belongs to the genus Gammacoronavirus in the subfamilyCoronavirinae, family Coronaviridae, order Nidovirales (de Grootet al., 2012). It has a single-stranded, positive-sense RNA genome ofapproximately 27.6 kilobases in length that encodes four structuralproteins: the nucleocapsid (N), membrane (M), envelope (E), andspike (S). Serotype and genotype classifications, which are usuallybased on features of the S1 part of the S protein gene, are used to

* Corresponding author at: Division of Avian Infectious Diseases, State KeyLaboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, theChinese Academy of Agricultural Sciences, Harbin 150001, People’s Republic ofChina. Fax: +86 451 82734181.

E-mail address: [email protected] (S. Liu).

http://dx.doi.org/10.1016/j.vetmic.2015.10.0030378-1135/ã 2015 Elsevier B.V. All rights reserved.

classify IBV strains (de Wit, 2000). Furthermore, recombinationmay be involved in the evolution of IBV, and it probably occurs atmany positions within a given genome during mixed infections(Lai and Cavanagh, 1997).

Historically, the Massachusetts (Mass)-type viruses werebelieved to be the first and only serotype found in the USA andother regions of the world. However, many IBV serotypes havearisen and disappeared in the poultry industry since then (Cooket al., 2012). The control of IB is mostly accomplished through theuse of live attenuated vaccines. The use of commercially producedMass-type modified live virus vaccines began in the 1950s and hascontinued to this day. This type of vaccines, such as the Massstrains M41 and H120, are the most commonly used around theworld because such vaccines have proven to be capable ofprotecting against a wide range of IBV strains (Cavanagh andGelb, 2008). However, the Mass-type of IBVs is constantly isolatedfrom chicken flocks with respiratory clinical signs.

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242 L. Chen et al. / Veterinary Microbiology 181 (2015) 241–251

In this study, the surveillance of IBVs was conducted on1717 chicken flocks suspected to be infected with IBV between2009 and 2013 in China. Twenty-nine Mass genotype IBVs wereisolated and, together with our previously isolated two strains, thegenomic and antigenic characteristics of these IBV isolates wereinvestigated to provide a better understanding of the emergence,circulation, evolution, and antigenicity of Mass-type IBVs inchicken flocks in China.

2. Material and methods

2.1. Eggs and chicks

Fertile White Leghorn specific pathogen-free (SPF) chicks wereobtained from the Harbin Veterinary Research Institute, as wereWhite Leghorn SPF chicken eggs. The birds were maintained inisolators with negative pressure, and food and water wereprovided ad libitum.

2.2. Samples, testing, and virus isolation

Samples of tissues and organs were obtained from 1717 chickenflocks with suspected IB clinical signs in China between 2011 and2013 that were vaccinated with the H120 vaccine (Table 1). Thesesamples were first screened for IBV by reverse transcription-polymerase chain reaction (RT-PCR) as previously described (Liuet al., 2009). Then the IBV-positive samples were used for virusisolation using 10-day-old embryonated SPF chicken eggs aspreviously described (Liu et al., 2009). Subsequently, a Massgenotype-specific RT-PCR assay targeting the IBV S1 gene (Cav-anagh et al., 1999) was conducted on the infected allantoic fluids,and only those viruses of the Mass genotype were used in thisstudy.

Table 1Characteristics of the IBVs included in the present studya.

IBV strain Year Province Day Vaccine used

ck/CH/LHLJ/091205 2009 Heilongjiang 20 H120

ck/CH/LHN/090909 2009 Henan 18 H120

ck/CH/LDL/110931 2011 Dalian 30 H120

ck/CH/LHB/110526 2011 Hebei 20 H120

ck/CH/LHB/110825 2011 Hebei 25 H120

ck/CH/LHB/111172 2011 Hebei 24 H120

ck/CH/LHB/111232 2011 Hebei 18 H120

ck/CH/LHB/111268 2011 Hebei 25 H120

ck/CH/LHLJ/110310 2011 Heilongjiang 6 H120

ck/CH/LHLJ/111050 2011 Heilongjiang 46 H120

ck/CH/LSD/110505 2011 Shandong 21 H120

ck/CH/LSD/110529 2011 Shandong 15 H120

ck/CH/LSD/110726 2011 Shandong 13 H120

ck/CH/LSD/111219 2011 Shandong 34 H120

ck/CH/LSD/111241 2011 Shandong 26 H120

ck/CH/LSD/1112150 2011 Shandong 23 H120

ck/CH/LDL/120557 2012 Dalian 14 H120

ck/CH/LHB/120403 2012 Hebei 45 H120

ck/CH/LHB/121024 2012 Hebei 25 H120

ck/CH/LHB/121040 2012 Hebei 25 H120

ck/CH/LHB/120749 2012 Hebei 23 H120

ck/CH/LJL/121059 2012 Jilin 35 H120

ck/CH/LSD/121228 2012 Shandong 20 H120

ck/CH/LHB/130573 2013 Hebei 24 H120

ck/CH/LHB/130598 2013 Hebei 18 H120

ck/CH/LHB/130642 2013 Hebei 25 H120

ck/CH/LHB/131118 2013 Hebei 20 H120

ck/CH/LHB/131132 2013 Hebei 15 H120

ck/CH/LHB/131142 2013 Hebei 23 H120

ck/CH/LHB/131143 2013 Hebei 15 H120

ck/CH/LHLJ/131216 2013 Heilongjiang 7 H120

a IBV strains ck/CH/LHLJ/091205 and ck/CH/LDL/090909 were isolated in 2009 (Sun

2.3. Total RNA isolation and complete genome sequencing and analysis

Total viral RNA was isolated from 200 ml of infected allantoicfluids using TRIzol reagent (Invitrogen, Carlsbad, CA, USA)according to the manufacturer’s instructions. The completegenomes of 31 Mass genotype of IBV strains were amplified andsequenced as previously described (Liu et al., 2013). The obtainednucleotide sequences were manually edited, assembled, andanalyzed using the Clustal W method available in the BioEditsoftware package (version 7.0.3.0., available at: http://www.mbio.ncsu.edu/bioEdit/bioedit) to produce the final sequences of theviral genomes. The S1 gene and genomes of our 31 IBV strains werecompared to those of the M41 and H120 strains and other19 reference strains available in GenBank. Genetic distances werecalculated using a maximum composite likelihood model with1000 bootstrap replicates as implemented in the MEGA 5.0program (Tamura et al., 2011).

To further identify the recombinant events, the BLASTNprogram (Liu et al., 2013) was used to search GenBank for IBVsequences that were homologous to those of the five IBV isolates,ck/CH/LHB/111172, ck/CH/LSD/111219, ck/CH/LHB/130598, ck/CH/LDL/110931, and ck/CH/LHB/130573. The Similarity Plot of thecomplete genomic sequences (Lole et al., 1999), the nucleotidesimilarities and phylogenetic trees for each deduced recombinantfragment were analyzed with the MEGA 5.0 program (Schierup andHein, 2000).

All of the 31 complete genomic sequences reported herein havebeen deposited in the National Center for Biotechnology Informa-tion's GenBank database, and the accession numbers are list inFig. 1A.

for immunization Organ used for virus isolation Type of chicken

Trachea BroilerTrachea BroilerProventriculus LayerKidney LayerKidney LayerKidney LayerProventriculus LayerProventriculus BroilerProventriculus + Kidney BroilerProventriculus LayerKidney BroilerProventriculus BroilerKidney BroilerKidney LayerProventriculus LayerProventriculus BroilerProventriculus LayerProventriculus BroilerTrachea LayerKidney BroilerProventriculus BroilerProventriculus BroilerKidney LayerProventriculus + Trachea BroilerProventriculus BroilerProventriculus BroilerProventriculus BroilerProventriculus BroilerProventriculus BroilerProventriculus BroilerProventriculus + Kidney Broiler

et al., 2011).

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Fig. 1. Phylogenetic trees constructed from the nucleotide sequences of the S1 subunit gene (A) and the complete genomes (B) of our 31 Mass genotype viruses and19 reference strains of infectious bronchitis viruses. The trees were computed using the neighbor-joining method. The significance of the tree topology was assessed by1000 bootstrapping calculations. Sequences of the viruses in this study are indicated in bold. GenBank accession numbers are indicated in parentheses.

L. Chen et al. / Veterinary Microbiology 181 (2015) 241–251 243

2.4. Virus cross-neutralization tests

The titers of the aforementioned five isolates and four deducedparental viruses (H120 and Connecticut vaccine strains and the ck/CH/LHB/100801 and tl/CH/LDT3/03 field strains) were determinedusing 10-day-old embryonated chicken eggs. The 50% embryoinfectious dose (EID50) was calculated by the method of Reed andMuench (1938). The b VN method with constant virus and dilutedserum was employed in SPF chicken embryos for serotyping.

2.5. Experimental design

One hundred and forty day-old SPF White Leghorn chicks weredivided into groups of ten birds (14 groups) and placed into moldedplastic isolators. Food and water were provided ad libitum. Chickensin groups 1, 3, 5, 7, 9, and 11 were inoculated with the H120 vaccineby oculonasal application at 1 day of age at a dose of log104 EID50/0.1 ml per chick. Birds in groups 2, 4, 6, 8, 10, 12, 13, and 14 weremock-inoculated with sterile allantoic fluid. Blood samples werecollected individually from each bird at days 4, 8,12,16, and 20 PI. At20 days PI, birds in groups 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10,and 11 and 12 were challenged by oculonasal application with 106

EID50/0.1 ml of IBV strains ck/CH/LHB/111172, ck/CH/LSD/111219,ck/CH/LHB/130598, ck/CH/LDL/110931, ck/CH/LHB/130573, andM41, respectively, while birds in group 13 were inoculated withthe H120 vaccine, and birds in group 14 were mock-inoculated withsterile allantoic fluid. The nasopharyngeal swabs were collectedindividually from each bird in all groups at days 5,10,15, and 20 PI todetect virus shedding by virus recovery and subsequent RT-PCRdetections as previously described (Liu et al., 2009). A positivesample was recorded if the specific lesions were observed and theRT-PCR amplification was positive. In addition, blood samples werealso collected individually from birds in all groups at days 4, 8,12,16,

and 20 post challenge. IBV-specific antibodies in serum sampleswere tested using a commercial total antibody ELISA (IDEXXCorporation, Westbrook, ME, USA) according to the manufacturer’sinstructions. The birds in each group were examined daily for signsof infection for 30 days post challenge.

3. Results

3.1. Virus detection, isolation, and typing

Using RT-PCR, we found 522 IBV-positive samples among thetissue and organ samples collected from 1717 chicken flocks thatwere suspected to be infected with IBV. Four hundred and eighteenIBVs were isolated from the 522 RT-PCR positive samples. Twenty-nine out of the 418 IBV isolates tested positive for the Massgenotype by primer-specific RT-PCR detection (Cavanagh et al.,1999). Most of the isolates were LX4-type. Other types of IBVisolates included tl/CH/LDT3/03-, TW I- and 4/91-like IBVs (datanot shown). The 29 Mass genotype IBV isolates and our twoprevious Mass type IBV strains (Sun et al., 2011) were used forsubsequent complete genomic sequencing.

3.2. Sequence comparison and phylogenetic analysis

As shown in Supplementary Table 1, the complete genomes of21 isolates each contained 27,630 nucleotides, excluding the poly-A tail at their 30 ends, which is similar to that of the H120 vaccinestrain. The genomes of two isolates had 27,473 nucleotides, whichis similar to that of the M41 strain. IBV isolates ck/CH/LHB/131132 and ck/CH/LDL/120557 contained 27,632 nucleotides each,with two insertions found in the 50 untranslated region (UTR)compared with that of the H120 strain. The remaining six isolates,ck/CH/LSD/111241, ck/CH/LSD/121228, ck/CH/LDL/110931, ck/CH/

Page 4: 2015 Molecular and antigenic characteristics of Massachusetts genotype infectious bronchitis coronavirus in China

Fig. 2. Recombination analysis of the ck/CH/LHB/111172 isolate. The similarity plot analysis is shown (A). Dotted lines show the deduced recombination breakpoints. Hollowarrows show the different fragments and their colors are the same as those of the parental viruses. Numbers show the nucleotide positions of the corresponding fragments inthe genome of the ck/CH/LHB/111172 isolate. Multiple sequence alignment of the predicted breakpoint and flanking sequences among the Mass 41, H120, ck/CH/LHB/111172,and ck/CH/LHB/100801 strains (B). Numbers to the right of each alignment show the nucleotide positions in the genome of each virus. The sequences of the ck/CH/LHB/111172 isolate are listed, and only nucleotides differing from those of the ck/CH/LHB/111172 isolate are depicted. The region where the template switches (breakpoints) havetaken place is in bold. Deleted nucleotides are indicated by a -. Phylogenetic analysis using the corresponding fragments among the Mass 41, H120, ck/CH/LHB/111172, ck/CH/

244 L. Chen et al. / Veterinary Microbiology 181 (2015) 241–251

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L. Chen et al. / Veterinary Microbiology 181 (2015) 241–251 245

LHB/130573, ck/CH/LHB/130598, and ck/CH/LSD/111219, contained27,604, 27,631, 27,582, 27,594, 27,609, and 27,654 nucleotides,respectively, with most of the differences (insertions/deletions)occurring in the 30 end of the genomes compared with those ofeither the H120 and M41 strains. Overall, the genomic organiza-tions of the 31 viruses are as follows: 50-UTR-Gene 1 (ORF1a, 1b)-S-Gene 3 (ORFs 3a, 3b, E)-M-Gene 5 (ORFs 5a, 5b)-N-UTR-30.

All 31 IBV strains in this study were of the Mass genotype basedon the S1 gene phylogenetic analysis (Fig. 1A). Of the 31 strains, theck/CH/LHB/130573 strain was not grouped with either the H120 orM41 strains based on the S1 gene phylogenetic analysis. Among the28 IBV strains that clustered with the H120 vaccine strain on thebasis of their S1 genes, 24 clustered in parallel with H120 in thephylogenetic tree constructed using the complete genomicsequences. Less than two amino acid substitutions were foundin the hypervariable regions I and II of S1 subunit betweenH120 and some of the H120-like IBV isolates (SupplementaryFig. 1). Two isolates, ck/CH/LHLJ/091205 and ck/CH/LSD/1112150,grouped with the M41 strain based on both S1 gene and thecomplete genomic sequences. The ck/CH/LDL/110931 was groupedwith M41 based on S1 gene, however, it was not grouped witheither the H120 or M41 strains based on the complete genomicsequences. The remaining four isolates, ck/CH/LHB/111172, ck/CH/LSD/111219, ck/CH/LHB/130598, and ck/CH/LHB/130573, did notcluster with either the H120 or M41 strains in the phylogenetic treeconstructed using the complete genome sequences (Fig. 1B). Thefive isolates were used in a subsequent recombination analysis.

3.3. Recombinant analysis

As shown in Supplementary Table 2, the sequences of fourisolates, ck/CH/LHB/111172, ck/CH/LHB/130598, ck/CH/LDL/110931,and ck/CH/LHB/130573, showed low similarity to those ofH120 from the S1 gene to the 30 UTR of the genome. The regionof the ck/CH/LHB/111172 isolate had a very similar sequence to thatof ck/CH/LHB/100801, which is a Taiwan-II (TW-II) genotype virus(Ma et al., 2012). For the ck/CH/LSD/111219 isolate, only thenucleotide sequence encoding the S protein showed identity withthat of the H120 strain, and, the remaining sequences showed aclose relationship with those of the DY07 strain, which belonged toLX4-type and was isolated in China in 2007 (He et al., 2012). Thenucleotide sequence from the M gene to the 30 end of the genomeof isolate ck/CH/LHB/130598 was closely related to that of the 4/91 strain. We found that the sequence from the S gene to the 30 endof the genome in isolate ck/CH/LDL/110931 was closely related tothe prototype Conn46 strain, while the region in isolate ck/CH/LHB/130573 shared a similar sequence identity with strain tl/CH/LDT3/03 (Liu et al., 2005).

SimPlot analysis confirmed the aforementioned results, and itwas clearly shown that the five IBV isolates arose fromrecombination events from a template switch (Figs. 2A, 3A, 4A,5A and 6A). Isolate ck/CH/LHB/111172 emerged from recombina-tion events between H120- and ck/CH/LHB/100801-like viruses,and two recombination breakpoints were observed, which werelocated at nucleotides 24143–24158 (at the 30 end of the 3b gene)and 25527–25535 (at the 50 end of the 5a gene) (Fig. 2B). Isolate ck/CH/LSD/111219 originated from multiple recombination eventsbetween H120- and DY07-like viruses and two breakpoints werelocated at nucleotides 18123–18133 (nsp14) and 24075–24079 (atthe 50 end of the 3b gene) (Fig. 3B). The ck/CH/LHB/130598 isolateoriginated from a recombination event between H120- and 4/91-like viruses, with the breakpoint located at nucleotides 25348–25358 (at the intergenic UTR between the M gene and gene 5)

LHB/100801, and TW2575/98 strains (C). Trees were constructed using the neighbor-joinck/CH/LHB/111172, ck/CH/LHB/100801, and TW2575/98 strains (D). The percentages of

(Fig. 4B). Isolates ck/CH/LDL/110931 and ck/CH/LHB/130573 origi-nated from H120- and Conn46- (Conn-) like viruses and H120- andtl/CH/LDT3/03-like viruses, with breakpoints at nucleotides21488–21528 and 2167221684, respectively (both at the 30 endof the S1 gene) (Figs. 5B and 6B ).

In addition, data from the phylogenetic trees (Figs. 2C, 3C, 4C,5C and 6C) and similarity analysis (Figs. 2D, 3D, 4D, 5D and 6D)using the corresponding gene fragments confirmed these resultsand strongly suggested that the five Mass genotype IBVs originatedfrom recombination events.

3.4. Antigenic properties of IBVs by virus cross-neutralization tests

As illustrated in Table 2, remarkable cross-neutralization ofH120 was observed with three isolates, ck/CH/LHB/111172, ck/CH/LSD/111219, and ck/CH/LHB/130598, and between the threeisolates themselves. In contrast, the three isolates did notneutralize the ck/CH/LHB/100801, Connecticut, and tl/CH/LDT3/03 strains. For the ck/CH/LDL/110931 and ck/CH/LHB/130573 iso-lates, virtually no cross-neutralization was observed with eitherMass serotype strains or other serotypes, which confirmed theabsence of an antigenic relationship between isolates ck/CH/LDL/110931 and ck/CH/LHB/130573 and the other strains investigatedin this study, indicating that isolates ck/CH/LDL/110931 and ck/CH/LHB/130573 represent two novel serotypes.

3.5. Protection provided by the H120 vaccine

As illustrated in Table 3, no overt disease was observed in chicksin the ck/CH/LDL/110931- and H120-inoculated groups. However,mild clinical signs, such as listlessness, huddling, and ruffledfeathers, were observed in some of the chicks inoculated withstrains ck/CH/LHB/111172, ck/CH/LSD/111219, ck/CH/LHB/130598,ck/CH/LHB/130573, and M41 from days 3 to 10 PI. The antibodieswere detected in birds of all groups (except the control group) after8 days PI. Challenge viruses were re-isolated from oropharyngealsamples of all or most of the chicks inoculated with our five strains,the H120 vaccine virus, and the M41 strain on days 5 and 10 PI.Sixty and forty percent of the chicks inoculated with strains ck/CH/LDL/110931 and ck/CH/LHB/130573, respectively, were still shed-ding viruses on day 15 PI.

In general, H120 vaccination provided complete clinicalprotection against challenge with the ck/CH/LHB/111172, ck/CH/LSD/111219, ck/CH/LHB/130598, ck/CH/LDL/110931, andM41 strains. However, 30% of vaccinated chicks showed clinicalsigns after challenge with strain ck/CH/LHB/130573. A significantreduction of challenge virus replication was achieved by vaccina-tion using H120 vaccines for the viruses ck/CH/LHB/111172, ck/CH/LSD/111219, ck/CH/LHB/130598, and M41, indicating thatH120 vaccination provided good protection. In contrast, most ofthe birds vaccinated with H120 vaccines were shedding virusesafter ck/CH/LDL/110931 and ck/CH/LHB/130573 challenge on days5 and 10, suggesting that H120 vaccination did not providesignificant protection against these two viruses.

4. Discussion

In this study, 418 IBVs were isolated using 10-day-old SPFchicken embryos from samples collected from 1717 chicken flocks(�24.5%) suspected to be infected with IBVs, and 29 out of the418 IBV isolates (�7%) belonged to the Mass genotype. The lowerproportion of the Mass genotype IBV in the samples collected inthis study was not surprising because Mass-based live IBV vaccines

ing method. Percentages of nucleotide sequence identity among the Mass 41, H120,nucleotide sequence identity of the corresponding gene fragments are indicated.

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Fig. 3. Recombination analysis of the ck/CH/LSD/111219 isolate. The methods used for recombination analysis are similar as those of isolate ck/CH/LHB/111172 in Fig. 2. Thesequences of Mass 41, H120, ck/CH/LSD/111219 and DY07 strains were compared and analyzed.

246 L. Chen et al. / Veterinary Microbiology 181 (2015) 241–251

are used extensively in China. It is generally believed that if a virusof this type is isolated from a Mass-vaccinated flock withrespiratory clinical signs, it is likely the reisolated vaccine virusand that some other serotype of IBV, even some other viruses must

be responsible for the respiratory disease. This was the case in thisstudy because 24 out of the 31 Mass-type IBVs appeared to beH120-like strains by genomic sequence analysis, although inser-tions were found in the genome of three out of these 24 H120-like

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Fig. 4. Recombination analysis of the ck/CH/LHB/130598 isolate. The methods used for recombination analysis are similar as those of isolate ck/CH/LHB/111172 in Fig. 2. Thesequences of Mass 41, H120, ck/CH/LHB/130598, and 4/91 strains were compared and analyzed.

L. Chen et al. / Veterinary Microbiology 181 (2015) 241–251 247

strains. However, the isolation of two M41-like viruses from twodiseased chicken flocks was somewhat unexpected, as all the flocks

had been vaccinated with Mass-based live vaccines. Molecularstudies have shown that an amino acid substitution at position

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Fig. 5. Recombination analysis of the ck/CH/LDL/110931 isolate. The methods used for recombination analysis are similar as those of isolate ck/CH/LHB/111172 in Fig. 2. Thesequences of Mass 41, H120, ck/CH/LDL/110931, and Conn46 1972 strains were compared and analyzed.

248 L. Chen et al. / Veterinary Microbiology 181 (2015) 241–251

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Fig. 6. Recombination analysis of the ck/CH/LHB/130573 isolate. The methods used for recombination analysis are similar as those of isolate ck/CH/LHB/111172 in Fig. 2. Thesequences of Mass 41, H120, ck/CH/LHB/130573, and Partridge/GD/S14/2003 strains were compared and analyzed.

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Table 2Titers of reciprocal b virus neutralization tests (diluted serum, constant virusnumbers).

Virus Serum

1 2 3 4 5 6 7 8 9

1. ck/CH/LHB/111172 256 160.9 128 146.4 3.5 1.4 1.8 <1 <12. ck/CH/LSD/111219 224 512 177 128 3.6 1.6 1.4 1:1.4 1.43. ck/CH/LHB/130598 159 211 164 158 1.7 2 <1 <1 1.14. H120 218 292 218 362 14 5.8 1.2 <1 1.15. ck/CH/LDL/110931 1.4 3.2 1.4 1.1 201 4.5 <1 <1 1.86. ck/CH/LHB/130573 <1 2.5 <1 <1 6.9 181 2 <1 11.27. ck/CH/LHB/100801 <1 2.4 1.6 <1 1.8 2.8 256 1.4 28. Connecticut 1.5 2.2 2.8 2.9 1.4 2.2 <1 299 2.49. tl/CH/LDT3/03 <1 1.4 <1 <1 11.2 21 1.6 <1 216

250 L. Chen et al. / Veterinary Microbiology 181 (2015) 241–251

63 of the S1 subunit of the spike protein in the M41 strain can resultin escape mutants (Cavanagh et al., 1988). In this study, we did notfind this substitution in the S1 subunit of our two M41-like isolates,indicating that the vaccination failure in the two flocks was not dueto a mutation in the S1 gene at this site.

In this study, by complete genomic sequence comparison,SimPlot analysis, and phylogenetic and similarity analysis usingthe corresponding gene fragments, our results clearly suggestedthat five of the 31 Mass genotype IBVs originated fromrecombination events. An H120-like virus was predicted to beone of the parental viruses for each of the recombinant viruses.This is not surprising because the H120 vaccine virus is commonlyused in China. In addition, the nucleotide sequences of S1 subunitof all the recombinant viruses was predicted to be derived from anH120-like virus because all the viruses were primarily screened byMass type-specific RT-PCR, the primers for which targeted theS1 subunit region (Cavanagh et al.,1999). Five IBV strains, includingDY07-, 4/91-, tl/CH/LDT3/03-, ck/CH/LHB/100801- and Conn-likeviruses, were predicted to be parental viruses for each of theisolates. In China, DY07-, tl/CH/LDT3/03- and ck/CH/LHB/100801-like viruses have been found to be prevalent in chicken flocks inrecent years (Liu et al., 2005; Han et al., 2011; Ma et al., 2012).Vaccine 4/91 was used in China for many years, although only a fewfield strains of this type were isolated. Interestingly, no Conn-likeviruses have been isolated in China thus far; however, similar to a

Table 3Results of vaccination-challenge tests using IBV strains ck/CH/LHB/111172, ck/CH/LSD/1

Groupa Dose (log10 EID50) Morbidity (%) Mortality (%)

1 V–C 6.0 0/10 (0%) 0/10 (0%)

2 C – 3/10 (30%) 0/10 (0%)

3 V–C 6.2 0/10 (0%) 0/10 (0%)

4 C – 1/10 (10%) 0/10 (0%)

5 V–C 6.2 0/10 (0%) 0/10 (0%)

6 C – 2/10 (20%) 0/10 (0%)

7 V–C 6.0 0/10 (0%) 0/10 (0%)

8 C – 0/10 (0%) 0/10 (0%)

9 V–C 6.0 3/10 (30%) 0/10 (0%)

10 C – 3/10 (30%) 0/10 (0%)

11 V–C 6.2 0/10 (0%) 0/10 (0%)

12 C – 6/10 (60%) 0/10 (0%)

13 – 0/10 (0%) 0/10 (0%)

14 – 0/10 (0%) 0/10 (0%)

a Birds in Groups 1, 3, 5, 7, 9, and 11 were vaccinated (V) with the H120 vaccine and130598, ck/CH/LDL/110931, ck/CH/LHB/130573, or M41. Birds in Groups 2, 4, 6, 8, 10, andCH/LHB/130598, ck/CH/LDL/110931, ck/CH/LHB/130573, or M41. Birds in Group 13 were oviruses and served as negative controls.

b Two procedures were used for virus recovery after vaccination/challenge as describednasopharyngeal swab samples were observed for lesions. Second, RT-PCR using a pair

allantoic fluid of the same eggs. The results from the two procedures were identical. Danumber of chicks used for attempted virus recovery after challenge.

C Days after vaccination/challenge.

previous report (Liu et al., 2014), the ck/CH/LDL/110931 isolate wasfound to be a naturally recombinant virus that arose from Conn-like and H120-like viruses.

The results in this study showed that the ck/CH/LHB/111172 andck/CH/LSD/111219 isolates experienced multiple recombinationevents and two breakpoints were identified. The recombinationevents from which the two viruses resulted can be explained bytwo models, similar to that of ck/CH/LZJ/111113 (Liu et al., 2013).For the remaining three viruses, ck/CH/LDL/110931, ck/CH/LHB/130573, and ck/CH/LHB/130598, only one breakpoint was identi-fied. The recombination event may have involved only twoparental viral strains with RNA replication initiating in an H120-like template of either negative or positive polarity, followed byswitching of the polymerase-nascent cRNA complex to a Conn-, tl/CH/LDT3/03-, and 4/91-like virus template, respectively.

It has already been demonstrated that there is a strongcorrelation between the S1 sequence, mainly in the hypervariableregion (HVR) 1/2, and protective relatedness values obtained in VNtests (Ladman et al., 2006). In the present study, this correlationwas confirmed by an analysis of the IBV strains ck/CH/LHB/111172,ck/CH/LSD/111219 and ck/CH/LHB/130598. However, two otherstrains, ck/CH/LDL/110931 and ck/CH/LHB/130573, although theyshared nearly the same sequences in the HVR1/2 regions with thatof the H120 strain, had different serotypes than the H120 strain andvaccination with the H120 strain did not provide protection againstchallenges with these two strains, as evaluated by tracheal virusshedding. It is believed that only a small number of amino acidschanges in the S1 domain are sufficient to change the serotype ofthe virus and affect cross-protection (Cavanagh et al., 1992). Wecompared the amino acid sequences of the S1 subunit and foundthat a mutation at nucleotide 118 resulted in a change from a Gresidue in Mass viruses to a V residue in the ck/CH/LDL/110931 andck/CH/LHB/130573 isolates. Alternatively, it was reported thatamino acid differences were responsible for the different second-ary structures of the S2 subunit of IBVs, which might result indifferent interactions between the S1 and S2 subunits and whichmight affect the conformation of the S1 subunit where serotype-specific epitopes are located, therefore accounting for serologicdifferences (Callison et al., 1999). We cannot conclude that thesubstitution in S1 and/or amino acid differences in the S2 subunits

11219, ck/CH/LHB/130598, ck/CH/LDL/110931, ck/CH/LHB/130573, and M41.

Virus recoveryb

5 dC 10 d 15 d 20 d

0/10 (0%) 0/10 (0%) – –

10/10 (100%) 10/10 (100%) 1/10 (10%) 1/10 (10%)1/10 (10%) 1/10 (10%) 0/10 (0%) –

10/10 (100%) 7/10 (70%) 1/10 (10%) 1/10 (10%)0/10 (0%) – – –

10/10 (100%) 7/10 (70%) 0/10 (0%) –

7/10 (70%) 5/10 (50%) 0/10 (0%) –

10/10 (100%) 8/10 (80%) 6/10 (60%) 0/10 (0%)8/10 (80%) 7/10 (70%) 1/10 (10%) 0/10 (0%)10/10 (100%) 9/10 (90%) 4/10 (40%) 2/10 (20%)1/10 (10%) 0/10 (0%) – –

10/10 (100%) 7/10 (70%) 2/10 (20%) 0/10(0%)10/10 (100%) 9/10 (90%) 2/10 (20%) 0/10 (0%)0/10 (0%) 0/10 (0%) 0/10 (0%) 0/10 (0%)

challenged (C) with IBV strains ck/CH/LHB/111172, ck/CH/LSD/111219, ck/CH/LHB/ 12 were only challenged with IBV strains ck/CH/LHB/111172, ck/CH/LSD/111219, ck/nly vaccinated with the H120 vaccine, and birds in Group 14 were not exposed to any

previously (Liu et al., 2014). First, embryos that had been inoculated with individualof oligonucleotide primers, N(�) and N(+), was conducted on RNA recovered fromta represent the number of chicks that showed a positive result after challenge/the

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L. Chen et al. / Veterinary Microbiology 181 (2015) 241–251 251

contributed to the serotype change of the two isolates in this study,as this will require further investigations using reverse genetic andvirus cross-neutralization tests.

Based on these findings, we conclude that extensive vaccinationwith modified live vaccines, such as H120, against differentserotypes, in association with the high recombination andmutation abilities of IBV, has led to the broad dissemination ofthe vaccine viruses and to the emergence of novel serotypes. Newstrategies to effectively control IBV infections in chickens should beconsidered, especially the use of inactivated and/or geneticallyengineered vaccines.

Competing interests

The authors declare that they have no competing interests.

Acknowledgement

This work was supported by grants from the China AgricultureResearch Systerm (No. CARS-41-K12), National “Twelfth Five-Year”Plan for Science & Technology Support (2015BAD12B03), andSpecial Fund for Agro-scientific Research in the Public Interest (No.201303033).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2015.10.003.

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